Aquaporin (AQP) water channels are believed to have a key role in the urinary concentrating mechanism and the pathophysiology of nephrogenic diabetes insipidus (NDI). The kidney expresses at least 4 AQPs: AQP1 in proximal tubule, TDLH, and vasa recta; AQP2 in the apical membrane of principal cells in the collecting duct; and AQP3 and AQP4 in the basolateral membrane of the same cells. Specific Aim 1 will define the role of AQPs in the urinary concentrating mechanism using transgenic knockout mice. Our lab developed the first transgenic mouse models to study water channel function-AQP1 and AQP4 knockout mice. These and AQP3 null mice will be used to define quantitatively the role of AQPs in renal water clearance. Renal function will be evaluated by urine/serum chemistries, isolated tubule and vasa recta microperfusion, and micropuncture. Specific Aim 2 will characterize the cellular defect in hereditary NDI and test a novel therapeutic strategy. Preliminary data indicate that AQP2 mutations cause NDI by heterogeneous mechanisms involving defective AQP2 water channel function, accelerated degradation, and defective intracellular processing with ER retention. Transfected cells expressing NDI-causing AQP2 mutants will be used to characterize AQP2 misfolding, degradation mechanisms, and interactions with molecular chaperones. A mouse model of NDI will be developed and used to evaluate the efficacy of chemical chaperones to correct defective AQP2 function in NDI. Specific Aim 3 will utilize novel biophysical methods to analyze specific aspects of AQP structure and function. Green fluorescent protein (GFP)-AQP chimeras will be used to define AQP mobility and association state in membranes and the cell biology of AQP2 trafficking. Methods will include fluorescence photobleaching recovery, energy transfer, and ratio imaging. Also, specific water/solute transporting properties (Pd, s s) of mammalian AQPs will be measured.